In 1899, Baeyer and Villiger reported on a reaction of cyclic ketones with peroxymonosulfuric acid to produce lactones (Chem Ber 32:3625–3633 (1899)). Since then, the Baeyer-Villiger (BV) reaction has been broadly used in organic synthesis. BV reactions are one of only a few methods available for cleaving specific carbon-carbon bonds under mild conditions, thereby converting ketones into esters (Walsh and Chen, Angew. Chem. Int. Ed. Engl 27:333–343 (1988)).
In the last several decades, the importance of minimizing environmental impact in industrial processes has catalyzed a trend whereby alternative methods are replacing established chemical techniques. In the arena of Baeyer-Villiger (BV) oxidations, considerable interest has focused on discovery of enantioselective versions of the Baeyer-Villiger oxidation that are not based on peracids. Enzymes, which are often enantioselective, are valued alternatives as renewable, biodegradable resources.
Many microbial Baeyer-Villiger monooxygenases enzymes (BVMOs), which convert ketones to esters or the corresponding lactones (cyclic esters) (Stewart, Curr. Org. Chem. 2:195–216 (1998), have been identified from both bacterial and fungal sources. In general, microbial BV reactions are carried out by monooxygenases (EC 1.14.13.x) which use O2 and either NADH or NADPH as a co-reductant. One of the oxygen atoms is incorporated into the lactone product between the carbonyl carbon and the flanking carbon while the other is used to oxidize the reduced NADPH producing H2O (Banerjee, A. In Stereosel, Biocatal.; Patel, R. N., Ed.; Marcel Dekker: New York, 2000; Chapter 29, pp 867–876). All known BVMOs have a flavin coenzyme which acts in the oxidation reaction; the predominant coenzyme form is flavin adenine dinucleotide cofactor (FAD).
The natural physiological role of most characterized BVMOs is degradation of compounds to permit utilization of smaller hydrocarbons and/or alcohols as sources of carbon and energy. As a result of this, BVMOs display remarkably broad substrate acceptance, high enantioselectivies, and great stereoselctivity and regioselectivity (Mihovilovic et al. J. Org. Chem. 66:733–738 (2001). Suitable substrates for the enzymes can be broadly classified as cyclic ketones, ketoterpenes, and steroids. However, few enzymes have been subjected to extensive biochemical characterization. Key studies in relation to each broad ketone substrate class are summarized below.
1. Cyclic ketones: Activity of cyclohexanone monooxygenase upon cyclic ketone substrates in Acinetobacter sp. NCIB 9871 has been studied extensively (reviewed in Stewart, Curr. Org. Chem. 2:195–216 (1998), Table 2; Walsh and Chen, Angew. Chem. Int. Ed. Engl 27:333–343 (1988), Tables 4–5). Specificity has also been biochemically analyzed in Brevibacterium sp. HCU (Brzostowicz et al., J. Bact. 182(15):4241–4248 (2000)).
2. Ketoterpenes: A monocyclic monoterpene ketone monooxygenase has been characterized from Rhodococcus erythropolis DCL14 (Van der Werf, J. Biochem. 347:693–701 (2000)). In addition to broad substrate specificity against ketoterpenes, the enzyme also has activity against substituted cyclohexanones.
3. Steroids: The steroid monooxygenase of Rhodococcus rhodochrous (Morii et al. J. Biochem 126:624–631 (1999)) is well characterized, both biochemically and by sequence data.
The genes and gene products listed above are useful for specific Baeyer-Villiger reactions targeted toward cyclic ketone, ketoterpene, or steroid compounds, however the enzymes are limited in their ability to predict other newly discovered proteins which would have similar activity.
The problem to be solved, therefore is to provide a suite of bacterial flavoprotein Baeyer-Villiger monooxygenase enzymes that can efficiently perform oxygenation reactions on cyclic ketones and ketoterpenes compounds. Identity of a suite of enzymes with this broad substrate acceptance would facilitate commercial applications of these enzymes and reduce efforts with respect to optimization of multiple enzymes for multiple reactions. Maximum efficiency is especially relevant today, when many enzymes are genetically engineered such that the enzyme is recombinantly expressed in a desirable host organism. Additionally, a collection of BVMO's with diverse amino acid sequences could be used to create a general predictive model based on amino acid sequence conservation of other BVMO enzymes. Finally, a broad class of BVMO's could also be used as basis for the in vitro evolution of novel enzymes.
Applicants have solved the stated problem by isolating several novel organisms with BVMO activity, identifying and characterizing BMVO genes, expressing these genes in microbial hosts, and demonstrating activity of the genes against a wide range of ketone substrates, including cyclic ketones and ketoterpenes. Several signature sequences have been identified, based on amino acid sequence alignments, which are characteristic of specific BVMO families and have diagnostic utility.